According to KR patent 0130100, production of biologically active substances through plant cell culture has more advantages than direct extraction from the plant. Plant cell culture is considered as an optimal method for continual production which is not influenced by environment and for solving the pending problems like destruction of ecology. Nail & Roberts (2004), however, indicated slow growth rate and low productivity of the plant cell culture for the secondary metabolite production. To solve this problem, there are studies of the optimization of the media, culture conditions, process and elicitation for higher productivity etc. (Zhong 2002). In the International patent WO93/17121, various media was used to culture diverse Taxus for the increase in cell growth rate and paclitaxel productivity. Based on the results of the experiments, elicitation conditions for paclitaxel mass production was indicated. Despite the improvements to the production of valuable secondary metabolites, variability is still a major issue for the production of paclitaxel from Taxus and other valuable substances from numerous plant systems.
Production of secondary metabolites through large scale plant cell culture is commercially possible only when there is a stable maintenance of rapid cell growth and high metabolite production during long term culture. The ability of the cell lines that could produce distinct metabolites are not stable which cause the cell lines to lose their initial productivity through subcultures; it is not too much to say that success and failure are depended on how we overcome these problems.
In plant cell culture, although the cells are derived from one plant, metabolite productivity of each cell line is different and unstable. Therefore, establishing the cell lines that have high productivity and genetic stability is most important than anything else.
Cell Lines Derived from Single Cells & and Multiple Cells
Plant cell lines derived from single cells have lower variability than the cell lines derived from multiple cells; this results in higher productivity. In preceding inventions, stem, root, seed, needle and leaf were used as the best explants for cell line induction. These stem, root, seed, needle and leaf are tissues that are composed of the cells with distinct functions and morphology. Callus, cell lines derived from these tissues is not of one kind. Therefore, there are limitations on the attempts to reduce the productivity variation of the callus derived from the tissues consisted of multiple cells.
Cell Aggregation
One of the distinguishing characteristics of plant cell culture is cell aggregation. According to the patent 0364478, diameter of the plant cell is 30-300 μm which is about 30 times bigger than the animal cell. Because plant cell walls have natural tendency to adhere together, it is not possible to obtain suspension which consists only of dispersed single cells. The proportion and the size of cell aggregates vary according to plant variety and the medium in which the culture is grown. Nail & Roberts indicated that cell aggregation leads to a difference in local environment between interior and exterior of the cells, which can result in culture heterogeneity and ultimately leads to changes in growth and metabolism.
The purpose of suspension culture is to obtain pure single cells. To accomplish this objective, filtration, maceration and protoplast culture by using enzyme were used. However, filtration and maceration do not provide complete pure single cells. Protoplast culture technique which eliminates the cell wall is the most reliable method for generating single cells, but the enzyme used for the protoplast culture cause cell wall damages or breakages that result in the change of cell physiology. Moreover, hydrophobic secondary metabolites such as paclitaxel can be stored in the cell wall, so the changes in the cell wall have profound relationship with productivity.
Also, cell aggregation has long been a major obstacle to the accurate measurement of cell growth by number and to biochemical assays to individual cells. According to Nail & Roberts (2004), if single cell culture is possible, it will readily provide faster information about the behavior of cell units in the culture such as biosynthesis, storage, and degradation etc. of secondary metabolites.
Dedifferentiation
The dedifferentiated cell line, which is callus, shows great variability in the production of secondary metabolites due to somaclonal variation. Callus derived from the permanent tissues such as leaves, stem, root and seed that are composed of the cells with distinct functions and morphology usually show dramatical changes even on slightly different micro-environments because it is a secondary meristem formed by dedifferentiation. Due to this sensitivity, Hirasuna et al. (1996) investigated to identify the cell culture conditions, especially initial cell density, subculture interval and temperature, and to maintain them as precisely as possible.
Scale Up
In order to produce secondary metabolites through plant cell culture for commercialization, scale up is essential. Bioreactor has been applied for mass production after many patents and articles were published, reporting about successful production of metabolites through cell culture in a laboratory scale. According to patent 0290004, application of bioreactor for mass production provides very different culture environment from the flask in a laboratory scale which results in the decrease in growth rate and productivity and change in the metabolites. When the bioreactor is applied for mass production, changes in growth rate, productivity and metabolites have become problems in commercialization of biologically active substances through cell culture. In the scale up of plant cell cultures, bioreactor which receives the air through exterior power or the bioreactor with impeller by considering the efficiency of the mixing and aeration are preferred. However, cell viability decreases abruptly in the bioreactor because plant cells are weak for shear. Therefore, a method to reduce shear is necessary. The cause of the shear sensitivity of the plant cell is explained by its large size, rigid cell wall, aggregation and extensive vacuolate (Yokoi, et al, 1993). To solve these problems in the bioreactor, a low shear generating bioreactor was investigated in the past by controlling its agitating speed and modifying the impeller type. However, it still bears negative results because the cell lines could not overcome the differences of the microenvironment.
Cryopreservation
Cryopreservation allows the long term cell maintenance by ceasing most of the metabolism of the cells in the extremely low temperature. It signifies the recovery of the cells without genetic, characteristics and biosynthetic variation after cryopreservation. By using cryopreservation, lost of the cells from contaminations could be eliminated and the genetic variation in the continuous cell lines could be minimized. In cGMP, the preservation of the cell lines for a long period is mandatory for the stable supply of raw materials. Usually, cultured animal cells could undergo cryopreservation for many years, but the similar cryopreservation technique is much more challenging for cultured plant cells. Cultured plant cells are heterogeneous and show diversity in physiology and morphology. Therefore, plant suspension cells require many processes for cryopreservation and inadequate cryopreservation could cause variability.
Conditioning Factors
Kim et al. (2000) demonstrated that cell division can be stimulated if some media from actively dividing cultures was added to the cultures that lost cell division ability. In the production of anthocyanin through rose suspension culture, the productivity increased when some media of strawberry suspension culture was added to the rose suspension culture. In this way, the factors that were produced and secreted from the cultured cells to stimulate the cell growth or the production of the secondary metabolites are called conditioning factors. Yet, these conditioning factors have not been identified concretely and there are only some understanding of conditioning factors acting as chemical signals for the cell growth and metabolite production. Also, there are few reports on the potent substances, such as phosphates and calmodium which could be considered as conditioning factors. Conditioning factors can be supplied through conditioned media or helper cells.
Perfusion Cultivation
Among the cell culture methods, there is a batch cultivation involving the inoculation of the cell and the media together in the beginning and no further nutrition supplementation. Also, there is a continuous cultivation, involving the supplementation of the new media as the spent media that contains metabolites is retrieved simultaneously at a consistent speed during the culture period for the prevention of nutrition depletion.
Batch cultivation is difficult in the commercial level due to its low productivity. Among the continuous culture methods, perfusion cultivation is receiving much attention these days. In perfusion culture, the cells are remained in the bioreactor, and new media is supplied as the spent media that contains metabolites is retrieved.
According to Zhang et al. (2000), elicitation is one of the most effective ways to promote the secondary metabolites production in cell culture. Elicitation encourages secondary metabolite synthesis, but it induces cell growth inhibition and the rapid decrease in the cell viability. Hence, secondary metabolite synthesis by elicitation could be maintained only for a short period and it is very limited. As Wang et al. (2001) presented, perfusion cultivation is a strategy to minimize these negative effects by elicitation and to maximize the productivity.
Wang et al. (2001) and Wu & Lin (2003) reported as follows. Secondary metabolites that are produced by elicitation are stored inside of the cell (vacuole or cell wall) or released outside of the cell (media). During the process of culture, releasing secondary metabolites from the cell and removing it from the media could bring easier purification and could diminish the feedback inhibition of biosynthesis and degradation and conversion of the products. Therefore, by retrieving the spent media and supplying with a new media, secretion of internal and external metabolites could extend the viability and biosynthesis of the cells. And it could remarkably increase the productivity.
Storage and the secretion of secondary metabolites showed great differences depending on the cell lines. Taxus media cell line (Wickremesinhe and Arteca 1994) did not excrete any. Consequently, establishing the cell line that has outstanding secretion ability is required.
Cambium Culture
Cambium is a lateral meristem that is located on the lateral side of the plant. In the gymnosperm and woody dicotyledon plants, there is a hypertrophic growth due to the continual activity of the cambium; as a result, giant plants having more than 11,000 years of the growth rings exist. In genetics, meristems could be classified as primary and secondary meristem. Primary meristem represents the meristem that forms during embryogenesis and participates in the plant growth after seed germination. Secondary meristem represents the meristem that is formed by dedifferentiation of the plant permanent tissue. Cambium is a primary meristem with meristematic continuity derived from the procambium without the intervene of the permanent tissue.
Growth of this primary meristem is indeterminate and could be continued if the conditions are given. Therefore, cambium culture has been used for rapid mass propagation of the cells.
In the preceding studies, cambium explants were prepared as follows: after the bark was peeled off, two longitudinal cuts, approximately 1 mm deep in order to reach the xylem, were made into the woody stem at an interval of 5 mm. They called these explants ‘cambium’, which was constituted of part of the phloem, cambium and a small chip of xylem (Jouira et al., 1998).
It is reasonable to say that cells which are induced by the method as mentioned above are not the sole origin of cambium, but of multiple tissues, which can be solemnly distinguished anatomically such as phloem, cambium and xylem. Thus, we could indicate that the method mentioned above is not the ideal technique to separate only the cambium elaborately from the various tissues that constitute the stems. A creative method to separate only the cambium from the various tissues of stems has been in demand.